The present application relates to an optical measuring device and method for identifying a sample such as minute particles, and more particularly to a technique for identifying the kind etc. of the sample by detecting fluorescence and scattered light generated from the sample irradiated with light having a specific wavelength.
In the case of identifying biological minute particles such as cells, microorganisms, and liposomes, an optical measuring method using flow cytometry (flow cytometer) is generally used (see Hiromitsu Nakauchi, supervisor, “Cell Engineering Separate Volume, Experimental Protocol Series, Flow Cytometry Jiyujizai,” Second Ed., Shujunsha Inc., (Aug. 31, 2006), for example). The flow cytometry is an analyzing method of individually identifying a plurality of minute particles flowing in a line in a channel by applying laser light having a specific wavelength to the minute particles and detecting fluorescence or scattered light generated from each minute particle irradiated with the laser light.
More specifically, a sample liquid containing a plurality of minute particles as an object to be measured and a sheath liquid flowing around the sample liquid form a laminar flow in a channel to line the minute particles contained in the sample liquid. In this condition, laser light is applied toward the channel, so that the minute particles are individually passed through the laser beam. At this time, each minute particle is excited by the laser light to generate fluorescence and/or scattered light, which are/is detected by using a photodetector such as a CCD (Charge Coupled Device) or a PMT (Photo-Multiplier Tube). The light detected by the photodetector is converted into an electrical signal and digitized to perform statistical analysis, thereby determining the kind, size, structure, etc. of each minute particle.
The scattered light to be measured in this flow cytometry is broadly classified into “forward scattered light” and “side scattered light.” The forward scattered light is light scattering at substantially the same angle as the incident angle of the laser light, and the side scattered light is light scattering in a direction perpendicular to the traveling direction of the laser light. For example, in the case that the sample as an object to be measured is a cell, it is widely known that the forward scattered light reflects information on the size of the cell and that the side scattered light reflects information on the form and internal structure of the cell. An analyzing device using this principle has already been developed (see Japanese Patent Laid-open Nos. 2006-230333 and 2007-263894, for example).
FIG. 11 is a schematic diagram showing the configuration of an existing optical measuring device 100 having a mechanism for measuring side scattered light. In the optical measuring device 100, exciting light 111 is applied from a light source 101 toward a detection area 109 of an analytical chip 102, so that scattered light is generated from a minute particle 110 in the detection area 109. A side scattered component 112 of this scattered light is detected on the upstream side of the analytical chip 102 in the traveling direction of the exciting light 111. More specifically, the side scattered component 112 generated from the minute particle 110 is passed through a condenser lens 103 and a pinhole 104 and next detected by a scattered light detector 105 such as a PMT.
On the other hand, a forward scattered component 113 of the scattered light is detected on the downstream side of the analytical chip 102 in the traveling direction of the exciting light 111 as in the case of detection of fluorescence. More specifically, the forward scattered component 113 generated from the minute particle 110 is condensed with fluorescence and the exciting light 111 by an objective lens 106. The exciting light 111 is next removed by an exciting light shielding mask 107. The forward scattered component 113 is next separated from the fluorescence by an LPF (Long Pass Filter) 108 and next detected by a detector (not shown).